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Effect of the O6 Substituent on Misincorporation Kinetics Catalyzed by DNA Polymerases at O6-Methylguanine and O6-Benzylguanine

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journal contribution
posted on 27.12.2001, 00:00 by Adrienne M. Woodside, F. Peter Guengerich
Misincorporation at a DNA−carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O6-alkylguanine. Replication of primer/templates containing guanine (G), O6-methylguanine (O6-MeG), or O6-benzylguanine (O6-BzG) was assessed using T7 DNA polymerase exo- (T7-) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7- and RT preferentially incorporated dTTP opposite O6-MeG and O6-BzG. The incorporation efficiencies (kcat/Km) were less for O6-BzG than O6-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase, i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O6-alkylG-containing substrates. Extension of the O6-BzG-containing duplexes was much more difficult for both polymerases as compared to O6-MeG, except when RT easily extended the O6-BzG:T base pair. The for binding of dCTP or dTTP to a RT•DNA complex containing O6-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA. The for a RT•DNA complex containing O6-BzG was 50-fold greater. In conclusion, the bulkier O6-BzG is a greater block to polymerization by T7- and RT than is O6-MeG, but some polymerization does occur with an O6-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O6-BzG-containing DNA, unlike the case of O6-MeG. These results and others regarding polymerase stalling opposite O6-MeG and O6-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039−1050].